Sensitive and Comprehensive Lipid Mediator Analysis using Advanced Scheduled MRM with Polarity Switching and QTRAP Enhanced Product Ion Scanning

Abstract

Using a QTRAP® 6500+ system coupled with an ExionLC™ System, we developed a targeted panel of 88 compounds for comprehensive profiling of lipid mediators and pathway markers. A Kinetex® Polar C18 column was used for LC based separation of lipid mediators, epimers and other isoelemental structures within a 20 min run time. Advanced scheduled sMRM was used to optimize scanning windows and dwell weighting. The negative mode lipid mediator panel includes pro‐inflammatory prostaglandins, leukotrienes and their primary metabolites, specialized pro‐resolving mediators (SPM) including resolvins, protectins, maresins, and lipoxins, biomarkers of ROS and NOS, and fatty acid and mono‐hydroxy fatty acid precursors. The positive mode panel includes cysteinyl leukotrienes, PAF, and maresin and protectin conjugates in tissue regeneration (MCTRs and PCTRs).Concentration curves were generated by injecting 3 replicate injections of 8 different concentrations of each lipid mediator standard. Excellent linearity is observed with r2 of at least 0.997 for all analytes. Excellent sensitivity was obtained for all lipid mediator species with LLOQ between 0.05 and 1 ng/mL and CV < 30%. The unique qualitative/quantitative QTRAP platform also enabled MRM triggered EPI experiments which provided high sensitivity MS/MS data for confirmation of low level analytes. Baseline separation of LXA4 and 15‐epi‐LXA4 well as RvD1 and 17‐epi‐RvD1 enables the differentiation of enzymatic pathway utilization for pro‐resolving lipid mediator production.